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Mixing Study Incubation Time

From Heather DeVries. Hi George! Do you know the reasoning behind using a 2-hour vs 1-hour incubation in mixing studies? We have always used 2 hours, and I wonder if it has to do with the heterogeneity of the antibodies we are trying to pick up. Our pathologist probably has a very good explanation, but I thought it would be interesting to hear from others on what they do and why. Thanks, Heather.

Hi, Heather, I have ordered a 2016 and a 2017 article on mixing studies, may have more to add tomorrow, but for now, the incubation period seems to be simply a matter of local preference. I once asked someone in the UAB special coagulation lab how long he incubated, his response was, “depends on how close we are to the end of the shift.” I’ve not seen any data that validate one interval over another, and I don’t know of any IgG immunoglobulins that require 2 hours to react. More on Wednesday, I hope.

Comments (4)
Mixing Studies
Apr 17, 2017 10:57am

Thank you for the comments!
Thank you for the comments! It makes sense to standardize our mixes to a two-hour incubation, since that is what we do for the VIII inhibitors.
Note from George, I agree, Heather, this makes sense. Nevertheless, we need a study that examines the inhibitor kinetics to settle the question.

Apr 4, 2017 10:03am

Heather, here is a comment I
Heather, here is a comment I received from Dr. Connie Miller, Consultant to the Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention:

“Hi George, We have not conducted any experiments on incubation time. In a chapter that I wrote, we said 1 hour. That should be adequate for most purposes. The rationale for using a 2-hour incubation is that that is the time used for factor VIII inhibitor assays. In contrast to other antibodies, factor VIII inhibitors are time-dependent. Testing a mix immediately and again at 1 or 2 hours may reveal time dependence. If the results are different, that suggests a specific factor VIII inhibitor, rather than some other type, such as a lupus anticoagulant. The choice of 1 hour or 2 hours may depend on the patient population being tested. Acquired factor VIII inhibitors are quite rare in pediatrics. Mixing studies should never be used as a substitute for an inhibitor assay in hemophilia patients, as mixing studies are not sensitive to low-titer inhibitors. If one is going to do an incubation anyway, doing a factor VIII level rather than a PTT in the patient and control mixes converts it to an inhibitor assay and allows calculation of units, without requiring much more time and effort.

Regards, Connie

Mar 23, 2017 3:43pm

Heather, the newly published
Heather, the newly published articles shed no further light on this subject. One wrote “1 or 2 hours,” the other failed to address incubation at all. I’m hoping for a response from a colleague who is an expert on the Nijmegen Bethesda assay, which as you know, specifies a 2-hour incubation as Mr. Crown writes above. My colleague may have conducted incubation time efficacy studies. Keep your fingers crossed.

Mar 22, 2017 4:08pm

I have said this before and I
I have said this before and I will say it again–there is an opportunity for someone to earn their doctorate by studying and standardizing mixing studies.
That being said, our procedure at St. Louis University Hospital coagulation laboratory for PT and aPTT mixing studies are 60 minutes at 37; no matter how close to the end of the shift we are.
If one wanted to argue that extending the incubation to 120 minutes would increase the sensitivity for time dependent inhibitors, i. e., factor inhibitors, that would make sense because we incubate our factor inhibitor assays for 2 hours.
Herb Crown, St. Louis University Hospital Coagulation Laboratory

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